PROJECT SUMMARY Most organisms employ an array of photoreceptors to detect their light environment. Arguably the most influential are the phytochromes (Phys), a diverse group essential for plant growth and development, and widely distributed in many bacterial, fungal, and algal genera. By reversible photointerconversion of their bilin chromophores between a red light- absorbing Pr state and a far-red light-absorbing Pfr state, Phys act as photoswitches in various signaling cascades responsive to light intensity, direction, duration, and spectral quality. Moreover, through the thermal reversion of Pfr back to Pr, some Phys sense temperature through enthalpic effects on the rate of this reaction, and possibly perceive time via the nighttime depletion of Pfr. The cumulative effects of this Pr/Pfr interconversion impact numerous physiological processes important to agriculture and the biology of harmful plant and human pathogens. In addition, their unique photochemistries have recently provided invaluable optogenetic tools, including novel fluorophores for tissue imaging, and engineered photoswitches that can regulate cellular events with remarkable temporal and spatial precision. Recently, we and others have made great strides in understanding how Phys signal through studies on the photosensing region. An emerging toggle model posits that a light-triggered isomerization of the bilin yields angstrom-scale rearrangements within the bilin-binding pocket that is ultimately transduced into large-scale conformational changes in the dimeric photoreceptor. While the model helps clarify gross changes required for endstate conversion, the intermediates of photoexcitation and ensuing structural changes necessary for a signaling-competent Pfr state are uncertain. It is also unclear how well the model applies to plant Phys given their distinctive modular architectures. The objective of this proposal is to complete this picture through continued structural and biochemical analyses of representative Phys in their Pr and Pfr states, and in combination with their downstream effectors. Specific aims are to: (1) use x-ray crystallography and cryo-electron microscopy to develop more comprehensive structures of plant and bacterial Phys, including models of full-length dimeric photoreceptors with theirs signal output modules; (2) define how Phys transduce the light signal through association with their downstream partners; (3) exploit serial femtosecond x-ray crystallography to structurally define the intermediates generated after photon absorption; (4) use steady-state and surface mapping methods to better understand the protein surface dynamics during photoconversion; and (5) appreciate how diversity within the plant Phy family is used to enhance thermal perception through the biochemical and structural analyses of the PhyB isoform that employs a predicted intrinsically disorder region at its N-terminus to sense temperature. Taken together, this project will provide an essential framework to better appreciate the structure, allosteric mechanism, and evolution of the Phy superfamily. Its anticipated results should help elucidate how microorganisms and plants sense light, temperature, and possibly time, which could have important ramifications for improving the agricultural performance of crop plants, understanding microbial ecosystems, controlling the life cycle of medically-relevant pathogens, and enhancing the application of Phys as optogenetic reagents.